YopN Is Required for Efficient Effector Translocation and Virulence in Yersinia pseudotuberculosis

Abstract

Type III secretion systems (T3SSs) are used by various Gram-negative pathogens to subvert the host defense by a host cell contact-dependent mechanism to secrete and translocate virulence effectors. While the effectors differ between pathogens and determine the pathogenic life style, the overall mechanism of secretion and translocation is conserved. T3SSs are regulated at multiple levels, and some secreted substrates have also been shown to function in regulation. In , one of the substrates, YopN, has long been known to function in the host cell contact-dependent regulation of the T3SS. Prior to contact, through its interaction with TyeA, YopN blocks secretion. Upon cell contact, TyeA dissociates from YopN, which is secreted by the T3SS, resulting in the induction of the system. YopN has also been shown to be translocated into target cells by a T3SS-dependent mechanism. However, no intracellular function has yet been assigned to YopN. The regulatory role of YopN involves the N-terminal and C-terminal parts, while less is known about the role of the central region of YopN. Here, we constructed different in-frame deletion mutants within the central region. The deletion of amino acids 76 to 181 resulted in an unaltered regulation of Yop expression and secretion but triggered reduced YopE and YopH translocation within the first 30 min after infection. As a consequence, this deletion mutant lost its ability to block phagocytosis by macrophages. In conclusion, we were able to differentiate the function of YopN in translocation and virulence from its function in regulation.

Identification of specific sequence motif of YopN ofYersinia pseudotuberculosisrequired for systemic infection

“…We have recently shown that the central region encompassing aa 76-181 is dispensable for the regulatory function of YopN but required for efficient translocation of effectors YopE and YopH in Yersinia. In accordance with this, a strain expressing YopN Δ76-181 -HA failed to block phagocytosis efficiently and was more readily internalized by macrophages [31]. In this study, we identified a putative coiled-coil domain encompassing aa 80-86 within the central region.…”

“…In a recent study we showed that the central region, encompassing aa residues 76-181 of YopN, with no previously assigned function, was dispensable for the regulatory function of YopN but translocated significantly lower amounts of YopH and YopE into HeLa cells compared to the wt strain. The significance of this finding was also corroborated by the finding that deletion of this region led to impaired blocking of phagocytosis by J774 macrophages [31]. The experiments in the previous study were done by expressing YopN Δ76-181 -HA from an arabinose inducible promoter in trans in a ΔyopN mutant background.…”

“…During the cloning procedure, we also added C-terminal hemagglutinin (HA) tags to each mutant to facilitate their detection by Western blot. We have previously shown that a C-terminal HA tag does not interfere with YopN function [31].…”

“…This phenotype is most likely the result of the lower levels on expression and/or stability of YopN Δ76-181 . When YopN Δ76-181 was expressed under full induction conditions in trans, expression levels were high enough to restore regulatory function [31], but when expressed in cis from the native promoter, the levels of YopN Δ76-181 were apparently too low for restoring regulation. In all, this strain was not suitable for in vivo infection studies as an attenuated phenotype in this case could be the result of the slightly impaired growth at 37°C and not necessarily be due to impaired function of YopN in the deletion mutant.…”

“…The results from the studies in Salmonella, Shigella and E. coli support a secretion hierarchy in these organisms where the translocators are secreted prior to the effectors [20,24,[26][27][28]. In Yersinia, however, ΔyopN mutants are not impaired for secretion of translocators [29][30][31]. One difficulty in assigning the role of YopN in virulence is that not only knock-out mutants but also mutants in the genes encoding SycN, YscB and TyeA become deregulated for T3SS expression/secretion and are unable to grow at host temperature, presumably because YopN stability is impaired and levels of YopN become very low [15,29,30,32,33].…”